Particles for Targeted Delivery and Uses in Managing Bleeding or Blood Clotting

ABSTRACT

This disclosure relates to microcapsule particles for targeted delivery of drugs. In certain embodiments, the particles comprise polyelectrolyte polymers, e.g., layers of anionic polymers and cationic polymers. In certain embodiments, the particles have a fibrinogen coating. In certain embodiments, the particles contain a polysaccharide core and/or a polysaccharide coating, encapsulating drugs, proteins, clotting agents, coagulation factors, or anticoagulants. In certain embodiments, this disclosure contemplates methods of using particles disclosed herein to prevent or reduce onset of or duration of bleeding. In certain embodiments, this disclosure contemplates methods of using particles disclosed herein to prevent or reduce onset of blood clotting.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.17/934,813 filed Sep. 23, 2022, which is a division of U.S. applicationSer. No. 16/074,643 filed Aug. 1, 2018 that granted as U.S. Pat. No.11,464,748 on Oct. 11, 2022, which is the National Stage ofInternational Application No. PCT/US2017/015872 filed Jan. 31, 2017,which claims the benefit of U.S. Provisional Application No. 62/289,642filed Feb. 1, 2016. The entirety of each of these applications is herebyincorporated by reference for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under HL121264 awardedby the National Institutes of Health and W81XWH-13-1-0495 awarded by theDepartment of Defense. The government has certain rights in theinvention.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED AS AN XML FILE VIA THEOFFICE ELECTRONIC FILING SYSTEM

The Sequence Listing associated with this application is provided in XMLformat and is hereby incorporated by reference into the specification.The name of the XML file containing the Sequence Listing is15095USDIV.xml. The XML file is 5 KB, was created on Sep. 22, 2022, andis being submitted electronically via the USPTO patent electronic filingsystem.

BACKGROUND

Mutations in the coagulation factor VIII gene result in a decreased ordefective coagulation factor (fVIII) protein that gives rise tohemophilia A, which is characterized by uncontrolled bleeding.Hemophilia B is similarly associated with a genetic defect incoagulation factor IX (fIX). Treatment of hemophilia A typically entailsrepeated intravenous infusion of either human plasma-derived orrecombinant fVIII product. Significant amounts of patients treated withfVIII replacement products develop neutralizing antibodies that renderfuture treatment ineffective. Thus, there is a need to identify improvedtherapies.

Voros et al. report nanoconstructs with immobilizing tissue plasminogenactivator molecules are capable of dissolving clots. Adv. Funct. Mater.2015, 25, 1709-1718. Korin et al. report shear-activatednanotherapeutics for drug targeting to obstructed blood vessels.Science. 2012, 337(6101):1453.

Hanafy et al. report the fabrication of homogenous CaCO₃ particles inassembling polyelectrolyte capsules. J. Basic Appl. Sci., 4 (2015),60-70. It also reports that these polyelectrolyte capsules are atemplate for encapsulation of cargo molecules either by usingco-precipitation or by loading cargo molecule after core removal.Poojari et al. report electrostatically mediated layer-by-layerassembled sorafenib nanoparticles. Colloids Surf B Biointerfaces. 2016,143:131-8.

U.S. Pat. No. 6,391,343 reports fibrinogen-coated particles fortherapeutic uses.

References cited herein are not an admission of prior art.

SUMMARY

This disclosure relates to microcapsule particles for targeted deliveryof drugs. In certain embodiments, the particles comprise polyelectrolytepolymers, e.g., layers of anionic polymers and cationic polymers. Incertain embodiments, the particles have a fibrinogen coating. In certainembodiments, the particles contain a polysaccharide core and/or apolysaccharide coating, encapsulating drugs, proteins, clotting agents,coagulation factors, or anticoagulants. In certain embodiments, thisdisclosure contemplates methods of using particles disclosed herein toprevent or reduce onset of or duration of bleeding. In certainembodiments, this disclosure contemplates methods of using particlesdisclosed herein to prevent or reduce onset of blood clotting.

In certain embodiments, this disclosure relates to a polymer-proteinmicrocapsule for targeted drug delivery of intravenously administeredblood clot regulating drugs. In certain embodiments, the microcapsulecomprises of at least four chemical regions comprising: a protein andpolymer layer on the exterior of the microcapsule shell, a microcapsuleshell comprised of biodegradable polyelectrolyte polymers, a separatepolymer layer on the inside of the microcapsule shell, and an aqueouscore containing a drug to be delivered. In certain embodiments, the drugto be delivered includes, but are not limited to, human or recombinantforms of Factor VIII, Factor VII, tissue plasminogen activator, orurokinase plasminogen activator.

In certain embodiments, this disclosure relates to particle having corecomprising drugs, proteins, clotting agents, coagulation factors, oranticoagulants encapsulated inside a coating comprising: a) a cationicpolymer or a polymer comprising cationic monomers; b) an anionic polymeror a polymer comprising anionic monomers; and c) an outer layer exposingfibrinogen or other protein on the outer surface.

In certain embodiments, the anionic polymer or polymer comprising theanionic monomers is under the outer layer. In certain embodiments, thecationic polymer or polymer comprising cationic monomers ispoly-L-lysine. In certain embodiments, the anionic polymer or polymercomprising anionic monomers is poly-L-glutamic acid.

In certain embodiments, the particles disclosed herein further comprisea polysaccharide layer such as a dextran layer. In certain embodiments,the cationic polymer and the anionic polymer is over the polysaccharidelayer.

In certain embodiments, the disclosure contemplates a particle ormicrocapsule for delivery of clot regulating drugs towards treatingblood-clotting disorders including, but not limited to, hemophilia A,hemophilia B, severe hemorrhage, heart attack, stroke, or thrombosis.

In certain embodiments, this disclosure relates to methods of treatingor preventing excessive bleeding comprising administering a particledisclosed herein to a subject in need thereof. In certain embodiments,the subject is diagnosed with hemophilia A or B or the subject isdiagnosed with acquired hemophilia or thrombocytopenia.

In certain embodiments, this disclosure relates to methods of producingparticle disclosed herein having core comprising a coagulation factorprotein encapsulated in a coating comprising: a) mixing carbonate saltsuch as sodium carbonate and a calcium salt such as calcium chlorideunder conditions such that a calcium carbonate core is formed; b) mixingthe calcium core and a polysaccharide under conditions such that apolysaccharide layer is formed over core to provide a polysaccharidelayered core; c) mixing the a polysaccharide layered core with ancationic polymer under conditions such that a polymer layer comprisingthe cationic polymer is formed providing a cationic polymer coated core;d) mixing the cationic polymer coated core with an anionic polymer underconditions such that an polymer layer comprising the anionic polymer isformed providing a cationic polymer layer and anionic polymer layercoated core; and e) mixing the cationic polymer layer and anionicpolymer layer coated core with fibrinogen under conditions such that anouter layer exposing fibrinogen on the outer surface is formed providinga fibrinogen coated particle.

In certain embodiments, the steps of both c) and d) wherein c) mixingthe a polysaccharide layered core with an cationic polymer underconditions such that a polymer layer comprising the cationic polymer isformed providing a cationic polymer coated core and d) mixing thecationic polymer coated core with an anionic polymer under conditionssuch that an polymer layer comprising the anionic polymer is formedproviding a cationic polymer layer and anionic polymer layer coatedcore, are repeated more than two, three, four, or five times.

In certain embodiments, the methods disclosed herein further comprisethe step of exposing the fibrinogen coated particle with a water solublechelating agent under conditions to remove the calcium ions in the coreof the particle, providing a fibrinogen coated particle depleted of thecalcium core, and mixing the fibrinogen coated particle depleted of thecalcium core with a drug under conditions such that the coagulationfactor is absorbed into the core providing a particle having corecomprising a coagulation factor protein encapsulated in a coatingcomprising anionic and cationic polymers.

In certain embodiments, the disclosure contemplates that a drug can beco-encapsulated in the calcium carbonate core by adding it into one ofthe salt solutions, e.g., before mixing the carbonate salt, e.g., sodiumcarbonate and calcium salt, e.g., calcium chloride together. In certainembodiments, the drug is then retained in the microcapsule when thecalcium carbonate core is removed

In certain embodiment, this disclosure contemplates pharmaceuticalcompositions comprising particles disclosed herein and pharmaceuticallyacceptable excipient. In certain embodiments, this disclosurecontemplates the production of a medicament comprising particlesdisclosed herein and uses for methods disclosed herein.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

FIG. 1 illustrates polyelectrolyte microcapsule production usinglayer-by-layer deposition of polyelectrolytes onto calcium carbonatecores. Microcapsules have been optimized to rupture under plateletcontraction and then deliver an encapsulated cargo.

FIG. 2 shows data on the zeta potential of the microcapsule duringpolyelectrolyte layer deposition indicating alternating surface chargeof the particles corresponding to the layer deposited, which confirmsthe layers were deposited.

FIG. 3 shows data on microcapsule loading. FVIII is loaded intomicrocapsule after production and purification steps are completed.

FIG. 4 shows data on the rate FVIII diffuses out of the microcapsules inplatelet poor plasma (PPP). There is still about 60% of FVIII left after3 hours, which is in the period of clot formation.

FIG. 5 shows data on fibrin formation in an in vitro blood vessel injurymodel. Shown are the normalized intensities of fibrin on thecollagen/tissue factor patch, which is a downstream indicator of theextent of clot formation, and FVIII efficacy on the patch. Allconditions used healthy patient blood to test components of themicrocapsule structure. The graph shows that there is an increase innormalized fibrin intensity when FVIII is delivered in the microcapsulescompared to systemic delivery. PEM MC refers to the polyelectrolytemicrocapsule. Fbn refers to fibrinogen.

FIG. 6 shows data fibrin formation in an in vitro blood vessel injurymodel with FVIII inhibitory antibody, MAb 2-76, to simulate hemophiliawith inhibitors. The data indicates that there is an increase innormalized fibrin intensity when FVIII is delivered in the microcapsulescompared to systemic delivery. PEM MC refers to the polyelectrolytemicrocapsule.

FIG. 7 shows data on the time required to form a blood clot for variousconditions. This set of experiments Advate™ (recombinant FVIII) is usedas the source. When Advate™ is delivered in the microcapsules comparedto systemically, there is a statistically significant decrease inclotting time. Furthermore, when blebbistatin is added, which inhibitsplatelet contraction; there is an increase in clotting time suggestingplatelet contraction is necessary for FVIII release.

FIG. 8A shows an image taken with a confocal microscope. Staticclot-like experiments were performed using fibrinogen, washed platelets,and microcapsules loaded with a model cargo. Microcapsule morphology wasmonitored when exposed to activated platelets in a fibrin network.Calcium, magnesium, and thrombin were added to initiate fibrin formationand platelet activation.

FIG. 8B shows an image taken with a confocal microscope indicating thatplatelet contractile forces are strong enough to cause microcapsulerupture and release of a model cargo. These microcapsules have beenoptimized to rupture under platelet contraction and then deliver anencapsulated cargo.

FIG. 9 shows a cross-sectional view of a microcapsule.

FIG. 10 shows a cross-sectional view of element 16 in FIG. 9 .

DETAILED DISCUSSION

Before the present disclosure is described in greater detail, it is tobe understood that this disclosure is not limited to particularembodiments described, and as such may, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present disclosure will be limited onlyby the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present disclosure, the preferredmethods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present disclosure is not entitled to antedate suchpublication by virtue of prior disclosure. Further, the dates ofpublication provided could be different from the actual publicationdates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentdisclosure. Any recited method can be carried out in the order of eventsrecited or in any other order that is logically possible.

Embodiments of the present disclosure will employ, unless otherwiseindicated, techniques of medicine, organic chemistry, biochemistry,molecular biology, pharmacology, and the like, which are within theskill of the art. Such techniques are explained fully in the literature.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. In this specification andin the claims that follow, reference will be made to a number of termsthat shall be defined to have the following meanings unless a contraryintention is apparent.

Prior to describing the various embodiments, the following definitionsare provided and should be used unless otherwise indicated.

“Cationic polymers” refer to macromolecules that are capable of bearingpositive charges in solutions at acidic or near neutral pH, which can beeither intrinsically present in the polymer backbone and/or in the sidechains. Typical cationic polymers possess quaternary amines or primary,secondary, or tertiary functional groups that can be protonated.Examples include natural cationic polymers such as gelatin.Semi-synthetic cationic polymers such as chitosan that has a repeatingamino group. The primary amino groups present on a polymer backboneprovides reactive sites for a variety of side-group attachments. Otherpolymers and polysaccharides such as dextrin, cyclodextrin, dextran, andcellulose can be modified to contain amine groups. Other cationicpolymers may be synthetic such as polyethylenimine, poly-L-lysine,poly-L-arginine, poly-L-histidine, poly(amidoamine),poly(amino-co-ester)s, poly[2-(N,N-dimethylamino)ethyl methacrylate] andcopolymers thereof.

“Anionic polymers” refer to macromolecules that are capable of bearingnegative charges in solutions at basic or near neutral pH, which can beintrinsically present in the polymer backbone and/or in the side chains.Typical anionic polymers possess carboxylic acid, sulfate and/orphosphate groups. Examples include polyacrylate, polymethacrylatepoly-L-glutamic acid, poly-L-aspartic acid, polyhydroquinone,polydibenzyl phosphate, polyvinyl sulfonate. Also contemplated arecopolymers such as acrylic or methacrylic acid that has been crosslinkedwith a di-functional monomer (e.g., divinylbenzene).

“Polysaccharides” refer to polymers having a sugar backbone. Examples ofpolysaccharides include dextrans, dextrins, chitosan, pullulans, andcelluloses. Dextrans refer to polysaccharides with molecular weightsthat are typically greater than 1,000 Dalton and have a linear backboneof repeating alpha-linked D-glucopyranosyl, such as isomaltose units.Dextrans are typically produced in bacteria. The molecular weight andspatial arrangement of dextrans depends on the microbial producingstrains and cultivation conditions. There are three classes of dextrandifferentiated by the structural features.

As used herein, the term “biodegradable” refers to a material that whentransplanted into an area of a subject, e.g., human, will be degraded mybiological mechanism such that the material will not persist in the areafor over a long period of time, e.g., material will be removed by thebody after a couple days or a week or month(s). In certain embodiments,this disclosure contemplates that the biodegradable material will not befound at the transplanted location after one day, two days, a week, amonth, six months, or a year.

As used herein, “subject” refers to any animal, preferably a humanpatient, livestock, or domestic pet.

As used herein, the terms “prevent” and “preventing” include theprevention of the recurrence, spread or onset. It is not intended thatthe present disclosure be limited to complete prevention. In someembodiments, the onset is delayed, or the severity is reduced.

As used herein, the terms “treat” and “treating” are not limited to thecase where the subject (e.g. patient) is cured and the disease iseradicated. Rather, embodiments of the present disclosure alsocontemplate treatment that merely reduces symptoms, and/or delaysdisease progression.

Polymer-Protein Microcapsules for Targeted Delivery of Blood ClotRegulating Drugs

Blood clot regulating drugs are administered topically for hemorrhage orintravenously for either clot promoting or clot busting treatments.Topical administration has a low risk of side effects but is ineffectivein treating hemorrhage from severe trauma or non-compressible injuries.Intravenous delivery, while more effective, is done systemically, whichincreases the risk of side effects. These side effects includehemorrhage from clot busting drugs or heart attack and stroke from clotpromoting drugs. Furthermore, patients suffering from bleeding disorderssuch as hemophilia A may form inhibitors to the drug, whichsignificantly decreases the drug's efficacy.

There exists a substantial need for targeted delivery systems of clotregulating drugs administered intravenously, so as the patient is onlyexposed to the drug strictly at the site wherein clot regulation isneeded, thereby avoiding side effects and inhibitor interactions.However, current targeted delivery technologies that have been developedare inappropriate for treating critical conditions in restrainedperiods. They either require external resources (magnets or lasers) totarget or deliver the drug, or if they have biochemical targeting anddelivery mechanisms, taking several days or even weeks to fully delivertheir drug payload. A targeted delivery technology for clot regulatingdrugs would leverage existing biochemical and biomechanical pathwayswithin the body to target and deliver the full dose of the drug withoutexternal equipment and within a short time frame. It is an object of theinstant disclosure to address these needs.

Certain embodiments disclosed herein provides for therapeutics that aredelivered systemically and become active or released at sites in bloodvessels that require blood clot regulation. This is based upon usingmicron size capsules as vehicles that will target specific cells/tissuesby interaction with naturally occurring pathways in the blood clottingmechanism such as the contractile force of platelets and their adhesionto fibrinogen.

This disclosure relates to a drug delivery approach to treat unregulatedblood clotting in cases where severe blood loss and/or unwanted clottingoccurs. This includes disorders such as, deep vein thrombosis, pulmonarythrombosis, Factor V Leiden Mutation, PT gene mutation, Protein C and Sdeficiency, Hemorrhagic Stroke, Severe Bleeding, Hemophilia A/B, vonWillebrands disease.

Fibrinogen is a glycoprotein in vertebrates that helps in the formationof blood clots. Fibrinogen is a soluble, large, and complexglycoprotein, 340 kDa in size, which is converted by thrombin intofibrin during blood clot formation. During normal blood coagulation, acoagulation cascade activates the zymogen prothrombin by converting itinto the serine protease thrombin. Thrombin then converts the solublefibrinogen into insoluble fibrin strands. These strands are thencross-linked by factor XIII to form a blood clot. Factor XIIa stabilizesfibrin further by incorporation of the fibrinolysis inhibitorsalpha-2-antiplasmin and TAFI (thrombin activatable fibrinolysisinhibitor, procarboxypeptidase B), and binding to several adhesiveproteins of various cells.

By displaying fibrinogen on the exterior of the drug delivery vehicles,the drug-loaded vehicles will integrate into the fibrin network of theforming clot. The mechanical force of the contracting clot caused byactivated platelets will rupture the vehicle and will thereby deliverthe drug solely to the site of the clot.

In certain embodiments, this disclosure relates to a polymer-proteinmicrocapsule for targeted drug delivery of intravenously administeredblood clot regulating drugs. In certain embodiments, the microcapsulecomprises of at least four chemical regions comprising: a protein andpolymer layer on the exterior of the microcapsule shell, a microcapsuleshell comprised of biodegradable polyelectrolyte polymers, a separatepolymer layer on the inside of the microcapsule shell, and an aqueouscore containing a drug to be delivered. In certain embodiments, the drugto be delivered includes, but are not limited to, human or recombinantforms of Factor VIII, Factor VII, tissue plasminogen activator, orurokinase plasminogen activator.

Particles disclosed herein show activity and effect in a clot-like invitro system using static fibrin networks with a high concentration ofactivated platelets. Only platelets located in the fibrin networkruptured and released the drug. Drug delivery from capsules occurs onlyin specific clot-like structures and is controlled by contractivemechanical action of activated platelets

In certain embodiments, this disclosure relates to a particle havingcore comprising a drug, protein, or coagulation factor encapsulated in acoating comprising: a) a cationic polymer or a polymer comprisingcationic monomers; b) an anionic polymer or a polymer comprising anionicmonomers; and c) an outer layer exposing fibrinogen on the outersurface.

In certain embodiments, the anionic polymer or polymer comprising theanionic monomers is under the outer layer. In certain embodiments, thecoagulation factor is fVIII. In certain embodiments, the cationicpolymer or polymer comprising cationic monomers is poly-L-lysine. Incertain embodiments, the anionic polymer or polymer comprising anionicmonomers is poly-L-glutamic acid.

In certain embodiments, the particles disclosed herein further comprisea polysaccharide layer such as a dextran layer. In certain embodiments,the cationic polymer and the anionic polymer is over the polysaccharidelayer.

In certain embodiments, this disclosure relates to a polymer-proteinmicrocapsule for intravenous targeted drug delivery of the encapsulateddrug. An illustrated embodiment is shown in FIGS. 1, 9, and 10 . FIG. 9shows a microcapsule 10 in the size range of 2 to 10 microns containingan aqueous core 12 comprising a drug. A polymer layer 14 encapsulatesthe core 12. The polymer layer 14 is further encapsulated by an element16. Element 16 functions as the microcapsule shell and is comprised ofseveral polyelectrolyte polymer layers of alternating charge. A layer 18coats the exterior of the microcapsule shell and consists of anegatively charged protein and a negatively charged polyelectrolyte.

As illustrated in FIG. 9 , the core 12 may comprise at least one drug invarying concentrations. The drug may be in a human or recombinant formof, but not limited to, Factor VIII, Factor VII, tissue plasminogenactivator, or urokinase plasminogen activator. The polymer layer 14surrounds the core 12 of the microcapsule. The layer consists of atleast one of the following polymers of varying size: dextran, chitosan,PEG or PEG-PLGA.

The layer 14 functions as a barrier between the core 12 and the element16 so that there are limited chemical or electrostatic interactionsbetween the drug retained in the core 12 and the polymers of element 16.

The polymer layer 14 is surrounded by the element 16 of themicrocapsule, which comprises several layers of polyelectrolytepolymers. The polyelectrolyte polymers are repetitively layered aroundlayer 14 with alternating charge. The element 16 of the microcapsulecomprises a minimum of 4 polyelectrolyte polymer layers, which mayeither be cross-linked through chemical means or held together throughelectrostatic interactions. The polyelectrolyte polymers may be, but arenot limited to, poly-L-lysine, poly-L-glutamic acid, poly-L-arginine,poly malic acid, or dextran sulfate of various sizes.

The layer 18 of the microcapsule surrounds element 16 and functions asthe exterior of the microcapsule. The layer 18 is comprised of theprotein, fibrinogen, and a negatively charged polyelectrolyte in variousconcentration ratios. The polyelectrolyte used in layer 18 may or maynot be used in element 16. The components of layer 18, that is to saythe protein and negative polyelectrolyte polymer, may or may not bechemically cross-linked together. Furthermore, the components of layer18 may or may not be chemically cross-linked to the components ofelement 16. The protein's biochemical activity of layer 18 is retained,such that platelets can adhere to the microcapsule exterior by bindingto the protein after intravenous administration.

In FIG. 10 there is shown the element 16 from FIG. 9 in more detailillustrating the alternating layers of polyelectrolyte. In oneembodiment of the invention, there may be four polyelectrolyte layerssuch that 20 may be comprised of the positive polyelectrolyte polymer,22 may be comprised of the negative polyelectrolyte polymer, 24 may becomprised of the positive polyelectrolyte polymer, and 26 may becomprised of the negative polyelectrolyte polymer. The positivepolyelectrolyte polymers of 20 and 24 may be the same or differentpolyelectrolyte polymers. The negative polyelectrolyte polymers of 22and 26 may be the same or different polyelectrolyte polymers. Otherembodiments of the invention may have more polyelectrolyte polymerlayers in element 16. Each polyelectrolyte polymer layer of element 16consists of at least one polyelectrolyte polymer of the same charge andmay or may not be chemically cross-linked to the other polyelectrolytepolymer within the same layer. The polyelectrolyte polymer layers ofalternating charge may or may not be chemically cross-linked to eachother such that, in one embodiment of the invention, polyelectrolytepolymer layer 20 may be chemically cross-linked to polyelectrolytepolymer layer 22. This may occur for all polyelectrolyte polymer layers,some of the polyelectrolyte polymer layers, or none of them at all.

The mechanical properties of element 16 are such that the microcapsulestructure will withstand forces experienced during fabrication, storage,and handling. However, the mechanical properties of element 16 are weakenough such that the microcapsule will rupture open upon contraction ofplatelets on the microcapsule.

The advantages of the present invention include, but are not limited to,targeted delivery and burst release of the encapsulated drug. Theprotein of layer 18 imparts the advantage of targeted delivery byallowing platelets to adhere to the microcapsule exterior. The adheredplatelets allow the microcapsule to be directed and adhered to areasrequiring blood clot regulation (target sites for the drug carried incore 12 within the microcapsule). The microcapsule is further adhered totarget sites by the fibrinogen protein of layer 18, which polymerizesinto the target site's fibrin network. Once the microcapsule is adheredto the target site, physical contraction of nearby platelets providessufficient mechanical force to rupture the microcapsule shell (element16) and release the encapsulated drug from core 12. Burst release of thedrug occurs because layer 14 physically shields the drug from theelectrostatic charge of element 16, allowing full exposure of the drugto the target site.

Blood Coagulation Factors

In certain embodiments, particles and microcapsules disclosed hereinencapsulate a drug that can be a coagulation factor. The blood clottingsystem is a proteolytic cascade. Blood clotting factors are present inthe plasma as a zymogen, in other words in an inactive form, which onactivation undergoes proteolytic cleavage to release the active factorform the precursor molecule. The ultimate goal is to produce thrombin.Thrombin converts fibrinogen into fibrin strands. These strands are thencross-linked by factor XIII to form a blood clot.

Fibrinogen, or Factor I, has an approximate molecular weight of 340 kDa.The purification of human plasma fibrinogen by chromatography isdescribed. See Kuyas et al., Thromb Haemost. 1990, 63(3):439-44, andDempfle et al. Thromb Res. 1987, 46(1):19-27.

Factor X is the first molecule of the common pathway and is activated bya complex of molecules containing activated factor IX (FIXa), factorVIII, calcium, and phospholipids, which are on the platelet surface.Factor VIII is activated by thrombin, and it facilitates the activationof factor X by FIXa. Factor VIII (fVIII) contains multiple domains(A1-A2-B-ap-A3-C1-C2) and circulates in blood in an inactivated formbound to von Willebrand factor (VWF). The C2 domain is involved withfVIII binding to VWF. Thrombin cleaves fVIII causing dissociation withVWF ultimately leading to fibrin formation through factor IX (fIX).

Congenital hemophilia A is associated with genetic mutations in thefVIII gene and results in impaired clotting due to lower than normallevels of circulating fVIII. Hemophilia B is similarly associated withgenetic mutations in the fIX gene.

A treatment option for a patient diagnosed with hemophilia A is theexogenous administration of recombinant fVIII sometimes referred to asfVIII replacement therapy. In some patients, this therapy can lead tothe development of antibodies that bind to the administered fVIIIprotein. Subsequently, the fVIII-antibody bound conjugates, typicallyreferred to as inhibitors, interfere with or retard the ability of fVIIIto cause blood clotting. Inhibitory autoantibodies also sometimes occurspontaneously in a subject that is not genetically at risk of havinghemophilia, termed acquired hemophilia. Inhibitory antibodies assays aretypically performed prior to exogenous fVIII treatment in order todetermine whether the anti-coagulant therapy will be effective.

A “Bethesda assay” has historically been used to quantitate theinhibitory strength the concentration of factor VIII binding antibodies.In the assay, serial dilutions of plasma from a patient, e.g., prior tohaving surgery, are prepared and each dilution is mixed with an equalvolume of normal plasma as a source of fVIII. After incubating for acouple hours, the activities of factor VIII in each of the dilutedmixtures are measured. Having antibody inhibitor concentrations thatprevent factor VIII clotting activity after multiple repeated dilutionsindicates a heightened risk of uncontrolled bleeding. Patients withinhibitor titers after about ten dilutions are felt to be unlikely torespond to exogenous fVIII infusions to stop bleeding. A Bethesda titeris defined as the reciprocal of the dilution that results in 50%inhibition of FVIII activity present in normal human plasma. A Bethesdatiter greater than 10 is considered the threshold of response to FVIIIreplacement therapy. Thus, in certain embodiments, this disclosurecontemplates that a subject to receive administrations of pharmaceuticalcompositions comprising particles disclosed herein is diagnosed with aBethesda titer of greater than 5, 6, 7, 8, 9, or 10.

In blood plasma, Factor VIII is usually complexed with another plasmaprotein, von Willebrand factor (vWF), which is present in plasma in alarge molar excess to Factor VIII and is believed to protect Factor VIIIfrom premature degradation. Another circulating plasma protein, albumin,may also play a role in stabilizing Factor VIII in vivo. Factor VIIIpreparations use of albumin and/or vWF to stabilize Factor VIII duringthe manufacturing process and during storage. In certain embodiments,this disclosure contemplates particles comprising Factor VIII in thecore in combination with albumin and/or vWF.

FVIII is a large glycoprotein containing the domain structureA1-A2-B-activation peptide (ap)-A3-C1-C2. Gitschier et al., Nature,1984, 312, 326-330. Factor VIII domain boundaries refer to the humanfVIII amino acid sequence numbering as follows; residues 1-19 (SignalSequence), 20-391 (A1), 392-759 (A2), 760-1667 (B), 1668-1708 (ap),1709-2038 (A3), 2039-2191 (C1) and 2192-2351 (C2). Gitschier et al.,Nature, 1984,312, 326-330. (SEQ ID NO: 1):

1 MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQSDL GELPVDARFP PRVPKSFPFN 61TSVVYKKTLF VEFTDHLFNI AKPRPPWMGL LGPTIQAEVY DTVVITLKNM ASHPVSLHAVGVSYWKASEG AEYDDQTSQR EKEDDKVFPG GSHTYVWQVL KENGPMASDP LCLTYSYLSHVDLVKDLNSG LIGALLVCRE GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRDAASARAWPKM HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL EGHTFLVRNH 301 RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME AYVKVDSCPE EPQLRMKNNEEAEDYDDDLT DSEMDVVRFD DDNSPSFIQI RSVAKKHPKT WVHYIAAEEE DWDYAPLVLA 421PDDRSYKSQY LNNGPQRIGR KYKKVRFMAY TDETFKTREA IQHESGILGP LLYGEVGDTLLIIFKNQASR PYNIYPHGIT DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGPTKSDPRCLTR YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR NVILFSVFDENRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHSINGYV FDSLQLSVCL HEVAYWYILSIGAQTDFLSV FFSGYTFKHK MVYEDTLTLF PFSGETVFMS MENPGLWILG CHNSDFRNRGMTALLKVSSC DKNTGDYYED SYEDISAYLL SKNNAIEPRS FSQNSRHPST ROKQFNATTI 781PENDIEKTDP WFAHRTPMPK IQNVSSSDLL MLLRQSPTPH GLSLSDLQEA KYETFSDDPSPGAIDSNNSL SEMTHFRPQL HHSGDMVFTP ESGLQLRLNE KLGTTAATEL KKLDFKVSSTSNNLISTIPS DNLAAGTDNT SSLGPPSMPV HYDSQLDTTL FGKKSSPLTE SGGPLSLSEENNDSKLLESG LMNSQESSWG KNVSSTESGR LFKGKRAHGP ALLTKDNALF KVSISLLKTN 1021KTSNNSATNR KTHIDGPSLL IENSPSVWQN ILESDTEFKK VTPLIHDRML MDKNATALRLNHMSNKTTSS KNMEMVQQKK EGPIPPDAQN PDMSFFKMLF LPESARWIQR THGKNSLNSGQGPSPKQLVS LGPEKSVEGQ NFLSEKNKVV VGKGEFTKDV GLKEMVFPSS RNLFLTNLDN 1201LHENNTHNQE KKIQEEIEKK ETLIQENVVL PQIHTVTGTK NFMKNLFLLS TRONVEGSYDGAYAPVLQDF RSLNDSTNRT KKHTAHFSKK GEEENLEGLG NQTKQIVEKY ACTTRISPNTSQQNFVTQRS KRALKQFRLP LEETELEKRI IVDDTSTQWS KNMKHLTPST LTQIDYNEKE 1381KGAITQSPLS DCLTRSHSIP QANRSPLPIA KVSSFPSIRP IYLTRVLFQD NSSHLPAASYRKKDSGVQES SHFLQGAKKN NLSLAILTLE MTGDQREVGS LGTSATNSVT YKKVENTVLPKPDLPKTSGK VELLPKVHIY QKDLFPTETS NGSPGHLDLV EGSLLQGTEG AIKWNEANRPGKVPFLRVAT ESSAKTPSKL LDPLAWDNHY GTQIPKEEWK SQEKSPEKTA FKKKDTILSLNACESNHAIA AINEGQNKPE IEVTWAKQGR TERLCSQNPP VLKRHQREIT RTTLQSDQEEIDYDDTISVE MKKEDFDIYD EDENQSPRSF QKKTRHYFIA AVERLWDYGM SSSPHVLRNRAQSGSVPQFK KVVFQEFTDG SFTQPLYRGE LNEHLGLLGP YIRAEVEDNI MVTFRNQASRPYSFYSSLIS YEEDQRQGAE PRKNFVKPNE TKTYFWKVQH HMAPTKDEFD CKAWAYFSDVDLEKDVHSGL IGPLLVCHTN TLNPAHGRQV TVQEFALFFT IFDETKSWYF TENMERNCRA 1921PCNIQMEDPT FKENYRFHAI NGYIMDTLPG LVMAQDQRIR WYLLSMGSNE NIHSIHFSGHVFTVRKKEEY KMALYNLYPG VFETVEMLPS KAGIWRVECL IGEHLHAGMS TLFLVYSNKCQTPLGMASGH IRDFQITASG QYGQWAPKLA RLHYSGSINA WSTKEPFSWI KVDLLAPMIIHGIKTQGARQ KFSSLYISQF IIMYSLDGKK WQTYRGNSTG TLMVFFGNVD SSGIKHNIFN 2161PPIIARYIRL HPTHYSIRST LRMELMGCDL NSCSMPLGME SKAISDAQIT ASSYFTNMFATWSPSKARLH LQGRSNAWRP QVNNPKEWLQ VDFQKTMKVT GVTTQGVKSL LTSMYVKEFLISSSQDGHQW TLFFQNGKVK VFQGNQDSFT PVVNSLDPPL LTRYLRIHPQ SWVHQIALRM 2341EVLGCEAQDL Y.

Before cell secretion, fVIII is cleaved at the B/ap-A3 domain junctioninto A1-A2-B (heavy chain) and ap-A3-C1-C2 (light chain) subunits. fVIIIcirculates in the plasma as an inactive heavy chain/light chainheterodimeric procofactor that is non-covalently bound to von Willebrandfactor. Proteolytic activation of fVIII by thrombin results fromcleavages at Arg-372 between the A1 and A2 domains, Arg-740 between theA2 and B domains, and Arg-1689 between the ap and A3 domains. Duringthis process, the covalent linkage between the A1 and A2 domains islost, and the B domain and 41-residue ap are released, producing aheterotrimeric, A1/A2/A3-C1-C2 subunit structure. See Doering et al. JBiol Chem. 2004, 279(8):6546-52. A number of functional B-domain-deletedrecombinant factor VIII proteins containing a linker with recognitionsequence for PACE/furin processing sequence, RHQR (SEQ ID NO: 2),substituted for the B-domain are known. See Sandberg et al. ThrombHaemost. 2001, 85(1):93-100 and Brown et al., Mol Ther Methods Clin Dev.2014, 1:14036.

Methods of Use

In certain embodiments, this disclosure contemplates methods of usingparticles disclosed herein to prevent or reduce onset of or duration ofbleeding. In certain embodiments, this disclosure contemplates methodsof using particles disclosed herein to prevent or reduce onset of bloodclotting. In certain embodiments, the disclosure contemplates a particleor microcapsule for delivery of clot regulating drugs towards treatingor preventing blood-clotting disorders including, but not limited to,hemophilia A, hemophilia B, severe hemorrhage, heart attack, stroke, orthrombosis.

In certain embodiments, the disclosure relates to methods of inducingblood clotting comprising administering an effective amount of aparticle as disclosed herein to a subject in need thereof. In certainembodiments, the subject is diagnosed with hemophilia A or B or acquiredhemophilia and/or unlikely to respond to exogenous fVIII infusions.

In certain embodiments, the methods are provided to increase the speedor strength of blood clot formation. In certain embodiments, thisdisclosure contemplates methods of using particles disclosed herein toprevent or reduce onset of or duration of bleeding.

In certain embodiments, the methods include controlling and/orpreventing bleeding episodes in adults and children (0-16 years) withhemophilia A. In certain embodiments, the methods include perioperativemanagement, e.g., administration prior to surgery or anesthesia, inadults and children (0-16 years) with hemophilia A.

In certain embodiments, the methods include routine prophylaxis toprevent or reduce the frequency of bleeding episodes in adults andchildren (0-16 years) with hemophilia A. For prevention of bleedingepisodes, doses between 20 to 40 International Units of Factor VIII perkg body weight every other day (3 to 4 times weekly) may be utilized.Alternatively, an every third day dosing regimen targeted to maintainFVIII trough levels ≥1% may be employed. In certain embodiments, themethods contemplate a subject under the age of 6, is administered dosesof 25 to 50 IU of Factor VIII per kg body weight 3 to 4 times weekly.

In certain embodiments, this disclosure contemplates therapeutic methodscomprising the step of administering (e.g., to inject or infuse)fibrinogen coated particles disclosed herein intravenously for thepurpose of decreasing bleeding time in thrombocytopenic subject.Thrombocytopenic subjects lack a sufficient concentration of plateletsthat are essential cellular elements responsible for hemostasis. In athrombocytopenic animal, the number of platelets is not sufficient toform a plug quickly. As a result, it takes a much longer time forbleeding to stop. It is anticipated that in patients about to undergosurgery with major blood loss, or in trauma patients such as soldierswounded in the battlefield, even though they have a “normal” plateletcount, an augmentation of the number of particles will decrease bloodloss and lead to shortened surgical time. Subjects at risk ofthrombocytopenic include those with aplastic anemia, cancer in the bonemarrow, such as leukemia, cirrhosis (liver scarring), folate deficiency,myelodysplastic syndrome, and a vitamin B12 deficiency.

In certain embodiments, the methods are provided to prevent, decreasethe speed, reduce or weaken blood clot formation. In certainembodiments, the disclosure relates to methods of preventing bloodclotting comprising administering an effective amount of a particledisclosed herein carrying or encapsulating or comprising immobilizingtissue plasminogen activator molecules (tPA) and/or other anti-clottingagent on the interior of the particle, e.g., warfarin (coumadin),acenocoumarol, phenprocoumon, atromentin, phenindione, a heparin,heparin tetrasaccharide, pentosan polysulfate, phosphomannopentanosesulfate, factor IIa (dabigatran) and factor Xa (rivaroxaban, apixabanand edoxaban), to a subject in need thereof.

Pharmaceutical Compositions

In certain embodiment, this disclosure contemplates pharmaceuticalcompositions comprising particles disclosed herein and pharmaceuticallyacceptable excipient. In certain embodiments, this disclosurecontemplates the production of a medicament comprising particlesdisclosed herein and uses for methods disclosed herein.

Pharmaceutical compositions typically comprise an effective amount ofparticles and a suitable pharmaceutical acceptable carrier. Thepreparations can be prepared in a manner known per se, which usuallyinvolves mixing the particles according to the disclosure with the oneor more pharmaceutically acceptable carriers, and, if desired, incombination with other pharmaceutical active compounds, when necessaryunder aseptic conditions. Reference is made to U.S. Pat. Nos. 6,372,778,6,369,086, 6,369,087 and 6,372,733 and the further references mentionedabove, as well as to the standard handbooks, such as the latest editionof Remington's Pharmaceutical Sciences.

In certain embodiments, the disclosure relates to pharmaceuticalcompositions comprising particles disclosed herein and apharmaceutically acceptable excipient. In certain embodiments, thecomposition is a pill or in a capsule or the composition is an aqueousbuffer, e.g., a pH between 6 and 8. In certain embodiments, thepharmaceutically acceptable excipient is selected from a filler,glidant, binder, disintegrant, lubricant, and saccharide. Optionally,the pharmaceutical composition further comprises a second clotting agentsuch as aminocaproic acid (ε-aminocaproic acid), tranexamic acid,fibrinogen, and vitamin K.

Compositions suitable for parenteral injection may comprisephysiologically acceptable sterile aqueous or nonaqueous solutions,dispersions, suspensions or emulsions, and sterile powders forreconstitution into sterile injectable solutions or dispersions.Examples of suitable aqueous and nonaqueous carriers, diluents solventsor vehicles include water, ethanol, polyols (propylene glycol,polyethylene glycol, glycerol, and the like), suitable mixtures thereof,vegetable (such as olive oil, sesame oil and viscoleo) and injectableorganic esters such as ethyl oleate.

Prevention of the action of microorganisms may be controlled by additionof any of various antibacterial and antifungal agents, example,parabens, chlorobutanol, phenol, sorbic acid, and the like. It may alsobe desirable to include isotonic agents, for example sugars, sodiumchloride, and the like. Prolonged absorption of the injectablepharmaceutical form can be brought about by the use of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Solid dosage forms for oral administration include capsules, tablets,pills, powders and granules. In such solid dosage forms, the particlesmay be admixed with at least one inert customary excipient (or carrier)such as sodium citrate or dicalcium phosphate or: (a) fillers orextenders, as for example, starches, lactose, sucrose, glucose, mannitoland silicic acid, (b) binders, as for example, carboxymethylcellulose,alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, (c)humectants, as for example, glycerol (d) disintegrating agents, as forexample, agar-agar, calcium carbonate, potato or tapioca starch, alginicacid, certain complex silicates, and sodium carbonate, (e) solutionretarders, as for example paraffin, (f) absorption accelerators, as forexample, quaternary ammonium compounds, (g) wetting agents, as forexample cetyl alcohol, and glycerol monostearate, (h) adsorbents, as forexample, kaolin and bentonite, and (i) lubricants, as for example, talc,calcium stearate, magnesium stearate, solid polyethylene glycols, sodiumlauryl sulfate, or mixtures thereof. In the case of capsules, tablets,and pills, the dosage forms may also comprise buffering agents.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirs. Inaddition to the particles, the liquid dosage forms may contain inertdiluents commonly used in the art, such as water or other solvents,solubilizing agents and emulsifiers, for example, ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,benzyl benzoate, propylene glycol, 1,3-butylene glycol,dimethylformamide, oils, in particular, cottonseed oil, groundnut oil,corn germ oil, olive oil, Viscoleo™, castor oil and sesame oil,glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan or mixtures of these substances, and the like.

In certain embodiments, production processes are contemplated which twocomponents, d particles disclosed herein and a pharmaceutical carrier,are provided already in a combined dry form ready to be reconstitutedtogether. In other embodiments, it is contemplated that particlesdisclosed herein and a pharmaceutical carrier are admixed to provide apharmaceutical composition.

Providing a pharmaceutic composition is possible in a one-step process,simply by adding a suitable pharmaceutically acceptable diluent to thecomposition in a container. In certain embodiments, the container ispreferably a syringe for administering the reconstituted pharmaceuticalcomposition after contact with the diluent. In certain embodiments, thecoated particles can be filled into a syringe, and the syringe can thenbe closed with the stopper. A diluent is used in an amount to achievethe desired end-concentration. The pharmaceutical composition maycontain other useful component, such as ions, buffers, excipients,stabilizers, etc.

A “dry” pharmaceutical composition typically has only a residual contentof moisture, which may approximately correspond to the moisture contentof comparable commercial products, for example, has about 12% moistureas a dry product. Usually, the dry pharmaceutical composition accordingto the present invention has a residual moisture content preferablybelow 10% moisture, more preferred below 5% moisture, especially below1% moisture. The pharmaceutical composition can also have lower moisturecontent, e.g., 0.1% or even below. In certain embodiments, thepharmaceutical composition is provided in dry in order to preventdegradation and enable storage stability.

A container can be any container suitable for housing (and storing)pharmaceutically compositions such as syringes, vials, tubes, etc. Thepharmaceutical composition may then preferably be applied via specificneedles of the syringe or via suitable catheters. A typical diluentcomprises water for injection, and NaCl (preferably 50 to 150 mM,especially 110 mM), CaCl₂) (preferably 10 to 80 mM, especially 40 mM),human albumin (preferably up to 2% w/w, especially 0.5% w/w), sodiumacetate (preferably 0 to 50 mM, especially 20 mM) and mannitol(preferably up to 10% w/w, especially 2% w/w). Preferably, the diluentcan also include a buffer or buffer system so as to buffer the pH of thereconstituted dry composition, preferably at a pH of 6.2 to 7.5,especially at pH of 6.9 to 7.1.

In certain embodiments, the diluent is provided in a separate container.This can preferably be a syringe. The diluent in the syringe can theneasily be applied to the container for reconstitution of the drycompositions. If the container is also a syringe, both syringes can befinished together in a pack. It is therefore preferred to provide thedry compositions in a syringe, which is finished with a diluent syringewith a pharmaceutically acceptable diluent for reconstituting, said dryand stable composition.

In certain embodiments, this disclosure contemplates a kit comprising apharmaceutical composition disclosed herein and a container with asuitable diluent. Further components of the kit may be instructions foruse, administration means, such as syringes, catheters, brushes, etc.(if the compositions are not already provided in the administrationmeans) or other components necessary for use in medical (surgical)practice, such as substitute needles or catheters, extra vials orfurther wound cover means. In certain embodiments, the kit comprises asyringe housing the dry and stable hemostatic composition and a syringecontaining the diluent (or provided to take up the diluent from anotherdiluent container).

Examples Microcapsule Fabrication

The production of microcapsules is illustrated in FIG. 1 . Calciumcarbonate cores used to template the fabrication of microcapsules bymixing equal volumes of 0.3 M calcium chloride and 0.3 M sodiumcarbonate for 30 seconds. The mixture was left to stand for 10 minutes,then washed with DI water 3× and dried over vacuum. For cores with anadditional calcium carbonate layer loaded with dextran, theabove-fabricated cores were dissolved in DI water at 80 mg/mL. To thestirring core solution, 1 mL of 0.3 M sodium carbonate and 1 mL of 0.3 Mcalcium chloride with 5 mg dextran or dextran-FITC were simultaneouslyadded. Stir was continued for 30 seconds, and then the solution was leftto stand for 10 minutes. Cores were washed 3 times with DI water anddried under vacuum.

To make the microcapsules, 2 mg/mL solutions of poly-1-lysine (PLL) andpoly-1-glutamic acid (PLG) were made with 0.5 M NaCl, pH 6.25.Polyelectrolyte layers were deposited onto the cores with alternatingcharge in a layer-by-layer fashion. In brief, a 2% w/v solution of coreswas dispersed in the PLL solution for 10 minutes. The cores were thenpelleted at 200 g for 5 minutes and washed 3 times with 0.5 M NaCl pH6.25 before the next layer was deposited. To deposit the fibrinogen onthe surface of the microcapsule, a 1:1 solution of human fibrinogen andPLG was used for deposition of the last polyelectrolyte layer. When thedesired number of layers was deposited, the cores were dispersed in a0.2 M EDTA solution at pH 6.25 for 30 minutes to remove the calciumcarbonate. The microcapsules were then washed and pelleted several timeswith 5 mM IVIES pH 6.25. Microcapsules were then stored at 4° C. untiluse.

To load FVIII into the microcapsules, the appropriate concentration ofFVIII was incubated with the microcapsules at a particle density of 1million/mL for 1 hour in pH 6.25 MES buffer. The microcapsules were thenwashed and pelleted several times to remove any unloaded FVIII.

Zeta Potential

Zeta potential of cores during PEM synthesis was measured with a MalvernZetasizer Nano Series™ (Malvern, UK). Samples were diluted with DI waterat a 1:9 ratio. Each sample was measured 3 times at 20 runs for eachmeasurement. See FIG. 2 .

FVIII Diffusion

FIG. 3 is a graph concerning loading optimization of FVIII into themicrocapsule. Data on FVIII diffusive release is shown in FIG. 4 .Fluorescent measurements were taken on a Cytation 5 Imaging Reader™ byBioTek (Winooski, VT) and were conducted in triplicate. Loading ofFVIII-RBITC in calcium carbonate cores was calculated by measuring thefluorescence intensity of FVIII-RBITC in the wash fraction during corefabrication.

Experiments measuring the diffusive release of FVIII-RBITC out ofcapsules were conducted in PPP at 37° C. and under 40 rpm agitation. Atappropriate time points, capsules were pelleted, and an aliquot of thesupernatant was removed to measure diffusive release of FVIII-RBITC. APPP aliquot of the same volume was added back to the capsule solution topreserve sample volume. Fluorescence intensity of the supernatant wasmeasured and compared to a standard curve to calculate concentration ofreleased FVIII-RBITC in the supernatant.

Static Fibrin Clots

Static fibrin clot experiments were fabricated by mixing fibrinogen, 1U/mL thrombin, 10 mM calcium, washed platelets stained with Cell MaskDeep Red, and microcapsules on a glass slides treated with Sigmacote™.Clots were formed at 37° C. and 60% humidity and imaged via confocallaser scanning microscopy using a Zeiss LSM 710 NLO system (Thornwood,NY).

Clotting on Collagen/TF Patch in Microfluidic Channels

Data for clotting studies are shown in FIGS. 5-7 . To form theperpendicular collagen/TF patch in the center of the microfluidicchannel, a PDMS-based straight channel with a width of 2 μm was bondedto a clean glass slide. Collagen (0.5 mg/mL) and TF (4 nM) in 0.01 Macetic acid was perfused through the channel and incubated at roomtemperature for 1.5 hours. The PDMS straight-channel was removed andslide was rinsed with DI water and dried with nitrogen. A straightchannel was cut into the silicone transfer tape with a width of 1.2 Oneside of the tape was adhered to a piece of clean PDMS. The remainingside was then adhered to the glass slide containing the collagen/TFstrip. The tape was aligned such that the collagen/TF strip wasperpendicular to and in the middle of the tape straight channel. Afteradherence to the coverslip, the glass was blocked with 5% BSA for 1hour.

WB was perfused at 5 μL/min for 10 minutes followed by PPP at 5 μL/minfor 30 minutes. Both WB and PPP were recalcified to 5 mM and contained 2mM Mg, CD41a-APC (platelet specific antibody), 59D8-AF488 (fibrinspecific antibody), and experiment condition (PBS, systemic FVIII, orFVIII in microcapsules). For experiments mimicking hemophilia withinhibitory antibodies, MAb 2-76 was added to both WB and PPP. Sampleswere kept at the same total volume. They also contained the same WB orPPP volumes.

The collagen patch was monitored over time via confocal laser scanningmicroscopy using a Zeiss LSM 700 System™ (Thornwood, NY). Videos wereconstructed by taking images every 10 seconds. Tile scans were taken ofthe entire patch after the experiment ended and used to measurefluorescence intensity of fibrin, platelets, and FVIII on the patch viaImage J.

Clot Formation Time

For well plate clots citrated whole blood was mixed with MAb 2-76 for 30minutes followed by addition of 5 mM Ca, 2 mM Mg, and 12 pM TF. Advate™or microcapsules loaded with Advate™ were added at the appropriateconcentration. Samples (50 uL) were loaded into wells and washed withPBS to remove soluble blood products at appropriate time points untilthe wash solution was clear. Samples were considered clotted when theclot covered the bottom of well and remained unchanged between timepoints.

1. A method of reducing blood clotting comprising administering aneffective amount of particles to a subject in need thereof, wherein theparticles have a core comprising tissue plasminogen activator (tPA)encapsulated in a coating comprising: a) a cationic polymer layer,wherein the cationic polymer layer comprises poly-L-lysine; b) ananionic polymer layer, wherein the anionic polymer layer comprisespoly-L-glutamic acid, and the cationic polymer layer is over apolysaccharide layer; c) an outer layer exposing fibrinogen on the outersurface, wherein the anionic polymer layer is under the outer layer; andwherein the polysaccharide is a dextran.
 2. The method of claim 1,wherein the subject is diagnosed heart attack.
 3. The method of claim 1,wherein the subject is diagnosed with stroke.
 4. The method of claim 1,wherein the subject is diagnosed with thrombosis.
 5. The method of claim1, wherein the subject is diagnosed with pulmonary thrombosis.
 6. Themethod of claim 1, wherein the subject is diagnosed with deep veinthrombosis.
 7. The method of claim 1, wherein the subject is diagnosedwith Factor V Leiden.
 8. The method of claim 1, wherein the tissueplasminogen activator is a human recombinant form.
 9. The method ofclaim 1, wherein the particles are administered intravenously.